1. Field of the Invention
The present invention relates to a method and device for reducing oxygen present in a confined space with a reduced oxidase with color formation. In particular, the present invention relates to a method and device which detects oxygen in the confined space by means of a blue color resulting from oxidation by the oxygen of the reduced oxidase. The method and device is particularly useful in the food, pharmaceutical and biological industries.
2. Description of Related Art
Indicators can be used to monitor a wide variety of biological and chemical reactions and to control fluctuations in the concentration of oxygen inside a packaged product. One particular use is for fresh and minimally processed packaged foods which require control of the internal environment in particular oxygen concentration to insure their freshness and safety.
Enzymes, including the blue oxidases, laccase and ascorbate oxidase, have been used in oxidized form in biosensors fundamentally to detect substances in aqueous solution. Laccase has been incorporated in gelatin membranes to determine glucose and phenol (Wollenberger, U., et al., Methods in Enzymol. vol 137, Part D. Academic Press, Inc. (1986)) and electrodes which reduce oxygen have been obtained by adsorption of laccase on carbonic carriers (Varfolomeev, S. D., Methods in Enzymol. Vol. 137, Part D. pp. 430-440, Academic Press, Inc. (1986)). Ascorbate oxidase has been immobilized by affinity chromatography for assay of ascorbic acid (Mattiasson, B, et al., Carbohydr. Res. 102:273 (1982)).
Japanese Patent Appln. JP3236766A describes the use of ascorbate oxidase, a laccase phenol oxidase and an ascorbate substrate to deoxygenate a food containing the ascorbic acid. Oxygen is removed by the reduced oxidase to prevent the deterioration of the food. British Patent Appln. No. 2022249 describes a method where ascorbic acid is determined by means of the oxygen consumed from the reaction mixture and produced by an ascorbate oxidase. Neither method relates to provide a colorimetrically detectable response from the oxidase.
There is a need for a colorimetrically detectable response to the presence of oxygen while reducing the oxygen in a confined space, particularly at low concentrations (10% by volume or less). There is particularly a need for a method using a separate device which detects the presence of oxygen in a confined space where the presence of oxygen may be deleterious to a material in the confined space.